The ability to detect the presence of a particular biological molecule or part of such a molecule at a specific location is an indispensable tool in Biological researches and in medicine. Presently, the most frequently used method of detection is to label the molecule with a radioactive isotope and detect the presence of the label by the radiation (usually beta emission) of the label isotope.
Isotopes that are reliable as labels materials emit high-energy electrons at rapid rate. That makes them unsuitable for use in many potential applications. This is especially true in situations where the label isotopes must be applied to living tissues. Furthermore highly radioactive materials are hazardous to handle and have short storage life.
It is therefore highly desirable to have labeling materials that are only slightly radioactive, thus having long storage life; or better yet, not radioactive at all.
There have been serious attempts to define non-radioactive labels. However none of them have yet proven as reliable or as sensitive as radioactive Phosphorus-32.
In some applications, it would be extremely useful to have distinguishable labels on different parts of biological systems, and be able to tell which part is present. An example of such an application is sequencing of DNA.
One of the essential steps in the sequencing of DNA is the use of an elctrophoresis gel column. In this step, the DNA fragments are placed in a reservoir at one end of a polyacrylamide column. An electric field (typically 1KV over a distance of 50 mm) causes the DNA fragments to migrate through electrophoresis towards the anode (at the other end of the column). As the molecules of the DNA fragments travel down the column, some molecules travel faster than others, molecules of different speeds concentrate in discrete bands. After a time period (typically 2 hours), the DNA fragments are spread over a column of discrete bands. Within each band are DNA fragments of a particular migration speed. If the DNA fragments had been labeled with some means of identification (for example radioactive isotopes or fluorescent dyes) then their presences can be detected.
One of the most common and most reliable isotope for labeling is Phosphorus-32 which emits beta radiation (electrons). DNA fragments that are labeled with P-32 are easily detected (for example by x-ray film).
In the case of DNA sequencing, there are four different Dideoxy chain-terminations at the ends of the fragments that need to be identified. P-32 alone can only label one of them. It is still common practice to run four parallel electrophoresis columns with P-32 labeling a different terminations in each column.
It would be extremely useful to be able to have distinguishable labels on different Dideoxy chain-terminations so a single lane gel can be used and the identities of the nucleotides can be determined in real time as the bands pass by a detector that has the capability of distinguishing the identity of the different labels.
There are other beta emitting isotopes besides P-32, but their radiations are spread over a wide spectrum, so one Beta emitter is difficult to distinguish from another.
Other distinguishable labels have been used in attempts on automatic DNA sequencing. One such example is the use of several fluorescent dyes that re-emit lights of distinguishable colors. But the dyes are large molecules, attaching such a label to a DNA fragment changes the migration rates, and that causes problems in identification.
The present invention provides a new possibility for labeling molecules in biological studies. The method exploits the response of atomic nuclei to Nuclear Magnetic Resonance (NMR). One of the advantages of the present invention is the possibility of using several distinguishable labels in a system.
The use of nuclear magnetic resonance (NMR) in biology and medicine has become commonplace since the advent of Magnetic Resonance Imaging (MRI) machines. These machines use NMR to detect presence of hydrogen nuclei (protons) in water and fatty tissues. Since MRI is less invasive than X-ray and can detect features that are not observable by X-ray, it has become an indispensable tool in many disciplines of medicine. MRI machines are very expensive. Part of the expense is the cost of the magnets needed to provide the needed field over a large volume (most MRI machines are big enough for people to enter into the magnet area), another part of the expense is the sophisticated electronics, including computers, to map out the protons.
For many biological applications, including automatic sequencing of DNA, neither a large detection area nor a high-resolution image is needed. For such applications, inexpensive permanent magnets can be used instead of large super-conducting magnets, and detection instruments compact enough to fit into small laboratories can become a reality.
Another common application of NMR is in spectroscopy. There the spatial resolution is not critical, but the NMR output is scanned over a wide spectrum at very high spectral resolution.
For many biological applications where isotopes with the suitable NMR properties are used as labels, successful detection of the label isotope requires neither the spatial resolution of MRI nor the spectral resolution of spectroscopy. It can therefore be a much simpler machine.
Technologies developed for MRI and for spectroscopy can obviously be "borrowed", when applicable, to improve the detection of NMR-active labels in biological molecules.
Whereas MRI uses almost exclusively hydrogen as the NMR-active isotope, and spectroscopy uses mostly hydrogen and carbon-13; there are many other isotopes that are suitable for used as NMR-active labels in biological applications.
For applications to automated DNA sequencig, it would be highly desirable to be able to simultaneous identify all four bases at the end of a DNA chain--Adenine, Guanine, Thymine, and Cytosine (A,G,T & C). The present invention provides a possibility for using the following combination of labels:
______________________________________ A labeled with P-31 only, G labeled with P-31 + H-3, T labeled with P-31 + F-19 and C labeled with P-31 + F-19 + H-3. ______________________________________
Reasons for the above choice are described in greater details below, in the "Detailed Descriptions" section.